Use of protease in industrial fields has a long history and has spread widely to various fields including detergents such as laundry detergents, fiber modifiers, leather treating agents, cosmetic compositions, bath additives, food modifiers, and pharmaceuticals. Of these, proteases for detergents are produced industrially in the largest amount. Known are, for example, Alcalase (trade mark; product of Novozymes), Savinase (trade mark; product of Novozymes), Maxacal (trade mark; product of Genencor), Blap (trade mark; product of Henkel), and KAP (product of Kao).
Proteases are incorporated into detergents for the purpose of degrading stains, which are composed mainly of proteins adhered to clothes. In practice, stains contain not only proteins but also plural components having, mixed therein, organic matters and inorganic matters such as lipids derived from sebum and solid particles. There is accordingly a demand for the development of detergents having detergency high enough to remove such a complex stain.
Finding several alkaline proteases capable of retaining caseinolytic activity even in the presence of a high concentration of fatty acids and exhibiting excellent detergency for removal of a complex stain containing both proteins and sebum, and having a molecular weight of about 43,000, the present inventors applied a patent (refer to Patent Literature 1) on them. These alkaline proteases are different in molecular weight, primary structure, enzymatic properties and markedly strong oxidant resistance from subtilisin which is a conventionally known serine protease derived from microorganisms belonging to the genus Bacillus so it is advocated that they should be classified into a new subtilisin sub-family (refer to Non-patent Literature 1).
The above-described alkaline proteases have caseinolytic activity even in the presence of a high concentration of fatty acids and exhibits excellent detergency for the removal of even a complex stain containing not only proteins but also sebum and the like. But its production amount is not sufficient for its production on an industrial scale. When a further improvement in detergency, as well as the production on an industrial scale, is taken into consideration, an alkaline protease having similar properties to those of the above-described alkaline proteases and having a more potent proteolytic capacity has been demanded.
Examples of the conventionally known method for enhancing secretion of a target protein (enzyme) include improvement by mutagenesis of a host strain (enzyme producing bacterium), and improvement of a gene encoding the enzyme or a gene controlling the expression of a gene encoding the enzyme. No improvement example permitting an increase in the secretion amount of subtilisin is however found.
On the other hand, for improving the proteolytic capacity, ordinarily employed is a method of altering a protease gene, thereby increasing proteolytic activity per mg of protein, that is, specific activity. There is a detailed report on protein engineering alteration for improving the specific activity of subtilisin (refer to Non-patent Literature 2, Non-patent Literature 3, Non-patent Literature 4, Non-patent Literature 5 and the like). Alterations so far reported showed an improvement in specific activity for certain synthetic peptides, but did not improve the activity toward natural substrates which is considered to have an influence on detergency.
With regards to an improvement in specific activity toward natural substrates, it is reported that proteolytic activity toward casein can be improved by replacing isoleucine at position 31 of subtilisin E with leucine (refer to Non-patent Literature 6). This case does not serve as a reference, because in the above-described alkaline protease, the corresponding amino acid is essentially leucine; and the above-described alkaline proteases are different in enzymatic properties from subtilisin having a molecular weight of about 28,000.
An object of the present invention is to provide an alkaline protease having a more potent proteolytic capacity, exhibiting excellent detergency for the removal of a complex stain, and has high secretion capacity.
Patent Literature 1: (International Publication No. 99/18218)
Non-patent Literature 1: (Saeki, et al., Biochem. Biophys. Res. Commun., 279, (2000), 313-319)
Non-patent Literature 2: (Wells, et al., Proc. Natl. Acad. Sci. USA., 84, (1987), 1219-1223)
Non-patent Literature 3: (Wells, et al., Proc. Natl. Acad. Sci. USA., 84, (1987), 5167-5171)
Non-patent Literature 4: (Taguchi, et al., Appl. Environ. Microbiol., 64, (1998), 492-495)
Non-patent Literature 5: (Takagi, et al., Protein Eng., 11, (1998), 1205-1210, Bryan, Biochim. Biophys. Acta, 1543, (2000), 203-222)
Non-patent Literature 6 (Takagi, et al., J. Biol. Chem., 36, (1988), 19592-19596)